PROJECT SUMMARY There is a critical gap in our understanding of mechanisms that underlie spontaneous preterm birth (SPTB) in non-Hispanic (NH) black women. Preterm delivery (<37 weeks gestation) is the leading cause of mortality among non-anomalous neonates; survivors are at increased risk for lifelong intellectual, physical, and social disabilities compared with their term counterparts. NH black women are twice as likely as women of other races to deliver preterm. 17-alpha hydroxyprogesterone caproate (17P) prevents recurrent SPTB in some women, but is less effective for NH black compared with NH white women. The reasons for this variable responsiveness are poorly understood, and represent a critical knowledge gap. The long-term goal of this research is to identify NH black women at risk for 17P non-response, provide them with alternate therapies, and thereby reduce the risk of recurrent SPTB. The objective here is to quantify the role of nitric oxide (NO) pathways in the pathophysiology of recurrent SPTB among NH black women receiving 17P. Our central hypothesis is that aberrations in the NO pathway predispose NH black women to 17P non-response for the recurrent SPTB prevention. This hypothesis is based on our preliminary data and published literature showing maternal genotype, maternal blood-derived and placental-derived proteomic profiles, and DNA methylation in the NO pathway differ among women destined to be 17P non-responders. Furthermore, our studies show strong race-disparity in NO pathway genes. The rationale for this work is that it will provide new insight and increased understanding into the pathophysiology and biologic mechanisms of non-response to 17P for SPTB prevention among NH black women, a group at disproportionately high risk of 17P treatment failure. The central hypothesis will be tested by pursuing three Aims: (1) Determine which genes in the NO pathway are differentially expressed in the mid-trimester among women destined to be 17P non-responders, (2) Establish which NO pathway genes display differential CpG methylation in the mid-trimester in 17P non-responders, and (3) Quantify conservation of gene expression and epigenetic markers between mother and offspring. These aims will be assessed using longitudinal samples from 300 NH black women. This approach is innovative, because this project will examine epigenetic and expression changes in maternal circulating blood and in fetal- derived tissues in response to 17P, shedding light on acute changes that occur in response to 17P. The proposed research is significant because as methylation and gene expression patterns are recognizable in the second trimester, they may provide the basis for development of diagnostic tests to identify women at risk for recurrent PTB despite 17P prophylaxis. This project is directly aligned with the mission of the NIMHD, and will provide immediate and sustained clinical and public health impact to reduce disparities in PTB outcomes in NH black women and infants, thereby reducing neonatal mortality and lifelong morbidity.